HIF‐1α: coordinates lymphangiogenesis during wound healing and in response to inflammation

Abstract

This study aimed to investigate the mechanisms that coordinate lymphangiogenesis. Using mouse models of lymphatic regeneration and inflammatory lymphangiogenesis, we explored the hypothesis that hypoxia inducible factor-α (HIF-1α) is a central regulator of lymphangiogenesis. We show that HIF-1α inhibition by small molecule inhibitors (YC-1 and 2-methyoxyestradiol) results in delayed lymphatic repair, decreased local vascular endothelial growth factor-C (VEGF-C) expression, reduced numbers of VEGF-C(+) cells, and reductions in inflammatory lymphangiogenesis. Using transgenic HIF-1α/luciferase mice to image HIF-1α expression in real time in addition to Western blot analysis and pimonidazole staining for cellular hypoxia, we demonstrate that hypoxia stabilizes HIF-1α during initial stages of wound repair (1-2 wk); whereas inflammation secondary to gradients of lymphatic fluid stasis stabilizes HIF-1α thereafter (3-6 wk). In addition, we show that CD4(+) cell-mediated inflammation is necessary for this response and regulates HIF-1α expression by macrophages, as CD4-deficient or CD4-depleted mice demonstrate 2-fold reductions in HIF-1α expression as compared to wild-types. In summary, we show that HIF-1α is a critical coordinator of lymphangiogenesis by regulating the expression of lymphangiogenic cytokines as part of an early response mechanism to hypoxia, inflammation, and lymphatic fluid stasis.

Adaptive Immune Responses Regulate the Pathophysiology of Lymphedema

“…Interestingly, and consistent with our previous observations, [14] we found that depletion of CD4+ cells markedly inhibited tissue fibrosis in the regions of the tail exposed to chronic lymphatic stasis as compared with control animals or for hypoxia inducible factor-1 alpha (HIF-1α) stabilization and regulation of VEGF-A/C expression in chronic lymphedema and in inflammatory lymphangiogenesis. [19] The findings of our current study are consistent with these results as we found that depletion of CD4+ but not CD8+ or CD25+ cells markedly increased lymphangiogenesis in the lymphedematous regions of the tail. Similarly, we found that depletion of CD4+ cells or inflammation in transgenic mice lacking CD4+ cells markedly increased lymphatic vessels density in draining lymph nodes and was associated with increased expression of VEGF-A and VEGF-C. Our findings complement and add to the work of Koh and colleagues since unlike the former study we show that CD4+ cells (rather than T cells in general) are important regulators of lymphangiogenesis during wound repair and that these effects likely involve regulation of both proand anti-lymphangiogenic pathways.…”

“…[16,17,19] Consistent with these observations, we found that IL4 neutralization for 6 weeks decreased the number of α-sma + capillary lymphatics compared with controls; however, this difference did not reach statistical significance ( Figure 6E, Supplemental Figure 3A). In contrast, animals treated with IL4mAb for 3 weeks after development of established lymphedema had significantly fewer α-sma + /LYVE-1 + capillary lymphatics (3.3-fold; p<0.001).…”

“…[19] To further test this hypothesis, we injected bleomycin intradermally into tails of wild type mice to induce fibrosis without skin/lymphatic excision. Figure 4C).…”

“…[12,16,17,19] Although it is clear that VEGF-C is necessary for lymphatic regeneration in most settings, it is also clear that this stimulus is not sufficient to restore lymphatic function in the context of lymphatic stasis, suggesting that other stimuli actively or passively inhibit this process. [40,41] These findings are consistent with previous reports from our lab and others demonstrating that inhibition of anti-lymphangiogenic cytokines can augment lymphatic function even in the context of normal or decreased VEGF-C expression.…”

“…Lymphatic vessel density was determined using Metamorph Scanner and Metamorph Offline software (Molecular Devices, Sunnyvale CA) as previously described. [19] Subcutaneous tissue thickness analysis was performed in histological cross-sections located 1.5 cm distal to the surgical site by blinded reviewers (n=6-8 per group). The distance from the basal layer of the epidermis to deep fascia was analyzed in 4 standardized regions per section.…”